CD4 is a surface glycoprotein primarily expressed on cells of the T lymphocyte lineage including a majority of thymocytes and a subset of peripheral T cells. Low levels of CD4 are also expressed by some non-lymphoid cells although the functional significance of such divergent cellular distribution is unknown. On mature T cells, CD4 serves a co-recognition function through interaction with MHC Class II molecules expressed in antigen presenting cells. CD4.sup.+ T cells constitute primarily the helper subset which regulates T and B cell functions during T-dependent responses to viral, bacterial, fungal and parasitic infections.
During the pathogenesis of autoimmune diseases, in particular when tolerance to self antigens breaks down, CD4+ T cells contribute to inflammatory responses which result in joint and tissue destruction. These processes are facilitated by the recruitment of inflammatory cells of the hematopoietic lineage, production of antibodies, inflammatory cytokines and mediators, and by the activation of killer cells.
Rheumatoid arthritis (RA), an inflammatory disease of the synovium, is one manifestation of an autoimmune phenomenon which results in erosion, deformity, and destruction of joints. Like most autoimmune diseases, the etiology of RA is not well defined. However, it is known that RA is characterized by elevated levels of activated CD4.sup.+ T lymphocytes in the affected joints. Currently there is no cure for RA. First line therapy for RA is designed to provide relief for RA symptoms and to improve functional abilities over the short term. Second and third line immunosuppressors and steroids such as azathioprine, methotrexate and prednisolone, targeted at the underlying disease, are administered in more severe cases and are either only mildly effective or exhibit unacceptable toxicity for chronic therapy. Also, they do not protect against joint destruction.
Apart from RA, CD4.sup.+ cells have also been implicated in other chronic conditions including psoriasis, insulin-dependent diabetes mellitus, systemic lupus erythematosus and inflammatory bowel diseases. Moreover, it is probable that CD4 expression may be involved in other autoimmune diseases.
Given the involvement of T cells in the development and maintenance of autoimmune diseases, immunosuppression has become an important treatment strategy. Available immunosuppressive drugs such as cyclosporin A have been used successfully for the treatment of transplant rejection. However, their toxic side effects renders them unacceptable for chronic therapy of autoimmune diseases.
Depletion of the entire T cell population, including the CD4.sup.+ subset, in clinical settings, has been accomplished by methods including thoracic duct drainage, total lymphoid irradiation and lymphopheresis, resulting in clinical improvement in some patients. Current strategies are, however, focused on more selective agents that block unwanted immune responses without causing solid organ toxicity or other major side effects. One way this potentially can be achieved is by selective removal or inactivation of disease mediating T cells with monoclonal antibodies (mAbs). mAbs to CD4 represent one such strategy. In animal models of autoimmunity and transplantation, anti-CD4 mAbs arrest or reverse disease progression when administered prophylactically or therapeutically. In addition, initial results from some clinical trials with anti-CD4 mAbs in RA, psoriasis, inflammatory bowel disease and systemic vasculitis have provided some preliminary evidence of potential therapeutic efficacy.
Essentially, the objective of anti-CD4 mAb therapy is to arrest the autodestructive activity of CD4.sup.+ cells, particularly during acute phases of autoimmune disorder. The ultimate therapeutic goal is to impose a state of immunological unresponsiveness (anergy) or long-term tolerance to the insulting antigens (or specific tissues) that sustain the underlying disease, without compromising normal host defenses against opportunistic infections. Apart from RA, CD4 mAbs may also be beneficial for the treatment of other autoimmune diseases, e.g., insulin-dependent diabetes mellitus, systemic lupus erythomatosis, psoriasis, inflammatory bowel disease, and multiple sclerosis.
Because of the potential importance of anti-CD4 mAbs as immunotherapeutics, numerous companies and research groups have reported anti-CD4 mAbs as potential therapeutic agents. For example, Centocor has reported an anti-CD4 mAb referred to as Centara which is a chimeric murine mAb to CD4. Further, Johnson & Johnson/Ortho has reported OKT-4a, an anti-CD4 mAb, which is a humanized murine mAb. Still further, Burroughs Wellcome has reported an anti-CD4 mAb which is a humanized rat mAb to CD4. Also, both Sandoz and MedImmune (in collaboration with Merck) have developed anti-CD4 murine-humanized mAbs specific to CD4. Still further, Becton Dickinson, Immunotech and Boehringer Mannheim have both developed anti-CD4 mAbs.
Apart from anti-CD4 mAbs, various immunomodulators and drugs have been disclosed to possess potential applicability for treatment of RA. Such immunomodulators and drugs include, e.g., cellular adhesion blockers, cytokine receptor blockers, immunotoxins and T cell receptor antagonists. Specific examples include gamma interferon, anti-ICAM-1 (a murine anti-CD54 mAb which blocks leukocyte trafficking, adhesion), Campath-1H (rat-humanized anti-CDw52 mAb) IL-1 receptor, cA2 (a TNF-alpha chimeric mAb), CDP 571 (anti-TNF mAb), anti-IL-2R (humanized-murine anti-CD25 mAb), SDZ CHH 380 (murine-human anti-CD7 mAb), DAB486 IL-2 (IL-2 fusion toxin, non-specific for CD4 and CD8 cells), Antril (IL-1RA), anti-TCP (mAb's and proteins which target T cell receptor subsets), and XomaZyme-CD5 (murine anti-CD5 toxin conjugate).
Also, other immunomodulators and immunosuppressors having potential application for treatment of autoimmune diseases include Rapamycin (oral immunosuppressive), Therafectin, Leflunomide (immunosuppressive prodrug), Tenidap (cytokine modulator/CO-inhibitor), IMM-125 and RS-61443 (an oral immunosuppressive).
As noted, numerous monoclonal antibodies to CD4 having potential therapeutic applications have been reported. For the most part, these antibodies comprise murine mAbs, chimeric or murine-humanized anti-CD4 mAbs.
Murine monoclonal antibodies have potential utility in the diagnosis of human disease as well as in clinical trials as therapeutics for treatment of both acute and chronic human diseases, including leukaemias, lymphomas, solid tumors (e.g., colon, breast, hepatic tumors), AIDS and autoimmune diseases. However, murine antibodies are disadvantageous because they often result in an immune antibody response in the host against the murine monoclonal antibodies.
Mouse/human chimeric antibodies have also been reported. These antibodies comprise the binding characteristics of the parental mouse antibody and effector functions associated with the human constant region. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,775; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No. 4,816,397, all of which are incorporated by reference herein. Generally, these chimeric antibodies are constructed in preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas (Nishman et al., 47 Cancer Research, 999 (1987)). The library is then screened for variable regions genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line.
However, while such chimeric antibodies have been used in human therapy, they also are subject to some problems. Similar to murine monoclonal antibodies, human recipients may produce antibodies against the chimeric antibody. This is disadvantageous to the efficacy of continued therapy with the chimeric antibody.
As an improvement to conventional chimeric antibodies, some researchers have disclosed methods for the production of human monoclonal antibodies which should not be subject to such problems. See, e.g., Erlich et al., 34 Clinical Chemistry, 1681 (1988); Erlich et al., 7 Hybridoma, 385 (1988); Erlich et al., 6 Hybridoma, 151 (1987), and Erlich et al., 1 Human Antibody Hybridomas, 23 (1990). These references also hypothesize that non-human primate antibodies, e.g., chimpanzee monoclonal antibodies, should be well tolerated in humans because of their structural similarity to human antibodies. However, the production of antibodies in humans has obvious ethical constraints.
Because human antibodies are non-immunogenic in Rhesus monkeys (i.e., do not induce an antibody response), Erlich et al. also predict that primate antibodies should be non-immunogenic in humans. Erlich et al. (Id.) indicate that the testing of antibodies in humans is unnecessary if a primate antibody has a constant region identical to that of a human immunoglobulin or, at least, a structure no more different from a human immunoglobulin than different human antibodies differ from each other. Thus, they suggest that chimpanzee antibodies may be useful in human therapy.
As an improvement to known chimeric antibodies which are often antigenic in humans, related applications U.S. Ser. No. 08/476,237, filed Jun. 7, 1995, Ser. No. 08/347,072, filed Jan. 25, 1995, and Ser. No. 07/912,212, filed Jul, 10, 1992, Ser. No. 07/856,281, filed Mar. 23, 1992, and Ser. No. 07/735,064, filed Jul. 25, 1991, all incorporated by reference herein, describe the manufacture of Old World monkey monoclonal antibodies and chimeric antibodies derived therefrom produced by recombinant methods which contain the variable domain of an Old World monkey antibody (e.g., baboon or macaque), fused to a cloned human, chimpanzee or other monkey constant region or other monkey framework regions. These applications in particular describe the manufacture of such Old World monkey and chimeric antibodies derived therefrom against human antigens as well as the use of such chimeric recombinant antibodies as immunotherapeutic agents for the treatment of human disease.
These applications are based on the surprising discovery that evolutionarily distant monkeys (e.g., baboon or macaque monkeys (including cynomolgus and Rhesus monkeys)), unlike chimpanzees, are not only sufficiently different from humans to allow antibodies against human antigens to be raised in these monkeys even to relatively conserved human antigens, e.g., CD4 and CD54, but are sufficiently similar to humans to have antibodies that are structurally similar to human antibodies, so that no host anti-antibody response when such monkey antibodies, or recombinant chimeric antibodies derived therefrom, are introduced into a human.
These applications disclose that unlike some prior antibodies used for human therapy, including known chimeric antibodies, such chimeric antibodies do not suffer from several drawbacks, e.g., 1) immunogenicity and induction of human anti-antibody (HAA) response upon repeated administration necessary to treat chronic conditions, 2) relatively short half-life compared to human antibodies, and 3) lack of effector functions with human cells or complement.
The lack of these drawbacks is a significant advantage for human therapy. For example, in the case of chronic human diseases, including autoimmune diseases, or any disease where prolonged administration of an antibody is necessary, one of the major obstacles to repetitive antibody therapy is the host response to the therapeutic antibody. HAA responses are often unpredictable from one patient to another. Also, such responses are predominately, though not exclusively, directed against the constant region of the antibody molecule, and once they occur they often preclude, or reduce the effectiveness of therapy with that antibody, or another antibody of the same isotype. The recombinant chimeric antibodies described in the above-referenced applications will circumvent this problem and allow for the generation of antibodies of the appropriate specificity and desired effector function, and their use in production of recombinant antibodies.
These recombinant antibodies generally include an appropriate portion of the variable region of an antibody derived from an immunized monkey, which is necessary for antigen binding, and the constant region of an antibody from a human or chimpanzee. Therefore, this allows for maintaining specificities and high affinities of the monkey monoclonal antibodies, and desired effector functions by the appropriate selection of human or chimpanzee constant region.
Several of these related applications exemplify in particular a monkey/human chimeric antibody with specificity for CD4, referred to as CE9.1, which contains the heavy and light chain variable domain of an anti-CD4 monoclonal antibody produced in a cynomolgus monkey and the human immunoglobulin light chain lambda constant region and the human immunoglobulin heavy chain gamma 1 constant region. This antibody possesses some T cell depletion activity, but which is lower in comparison to previous CD4 monoclonal antibodies. However, it is desirable to produce antibodies which possess less or which are devoid of T cell depleting activity because this would potentially enhance their therapeutic potential.
These applications further describe preferred vector systems for the production of such chimeric antibodies, in particular TCAE 5.2 and TCAE 6 which comprise the following:
1) Four transcriptional cassettes in tandem order:
(a) a human immunoglobulin light chain constant region. In TCAE 5.2 this is the human immunoglobulin Kappa light chain constant region (Kabat numbering amino acids 108-214, allotype Km 3) and in TCAE 6 the human immunoglobulin light chain lambda constant region (Kabat numbering amino acids 108-215, genotype Oz minus, Mcg minus, Ke minus allotype). PA1 (b) a human immunoglobulin heavy chain constant region; in both constructs the human immunoglobulin heavy chain is a gamma/constant region (Kabat numbering amino acids 114-478 allotype Gm1a, Gm 12). PA1 (c) DHFR; containing its own eukaryotic promoter and polyadenylation region; and PA1 (d) NEO; also containing its own eukaryotic promoter and polyadenylation region.
2) The human immunoglobulin light and heavy chain cassettes contain synthetic signal sequences for secretion of the immunoglobulin chains; and
3) The human immunoglobulin light and heavy chain cassettes contain specific DNA links which allow for the insertion of light and heavy immunoglobulin variable regions which maintain the translational reading frame and do not alter the amino acids normally found in immunoglobulin chains.
However, notwithstanding what has been previously described, there still exists a need in the art for improved antibodies which are specific to CD4, which possess low antigenicity in humans which may be used therapeutically, e.g., for the treatment of autoimmune diseases such as rheumatoid arthritis. In particular, there is a need for producing anti-CD4 antibodies which exhibit improved properties, e.g., longer half-life and/or which substantially lack or are devoid of depleting activity.